Method for modifying plant growth characteristics

ABSTRACT

The present invention concerns a method for modifying plant growth characteristics by modifying expression in a plant of a nucleic acid encoding a methionine aminopeptidase (MAP protein) and/or by modifying level and/or activity in a plant of a MAP protein. The invention also relates to transgenic plants having modified growth characteristics, which plants have modified expression of a nucleic acid encoding a MAP protein. Particularly the present invention discloses a method to increase yield of a plant, preferably in a cereal such as rice or corn.

The present invention concerns a method for modifying plant growthcharacteristics. More specifically, the present invention concerns amethod for modifying plant growth characteristics by modifyingexpression of a nucleic acid encoding a methionine aminopeptidase (MAPprotein) and/or by modifying level and/or activity of a MAP protein in aplant. The present invention also concerns plants having modifiedexpression of a nucleic acid encoding a MAP protein and/or modifiedlevel and/or activity of a MAP protein, which plants have modifiedgrowth characteristics relative to corresponding wild type plants.

Given the ever-increasing world population, it remains a major goal ofagricultural research to improve the efficiency of agriculture.Conventional means for crop and horticultural improvements utiliseselective breeding techniques to identify plants having desirablecharacteristics. However, such selective breeding techniques haveseveral drawbacks, namely that these techniques are typically labourintensive and result in plants that often contain heterogeneous geneticcomponents that may not always result in the desirable trait beingpassed on from parent plants. In contrast, advances in molecular biologyhave allowed mankind to more precisely manipulate the germplasm ofplants. Genetic engineering of plants entails the isolation andmanipulation of genetic material (typically in the form of DNA or RNA)and the subsequent introduction of that genetic material into a plant.Such technology has led to the development of plants having variousimproved economic, agronomic or horticultural traits, for exampleimproved yield.

The ability to influence one or more of the plant growthcharacteristics, would have many applications in areas such as cropenhancement, plant breeding, production of ornamental plants,arboriculture, horticulture, forestry, production of algae or plants(for use as bioreactors for example, for the production ofpharmaceuticals such as antibodies or vaccines, or for the bioconversionof organic waste, or for use as fuel in the case of high-yielding algaeand plants).

It has now been found that modifying expression in a plant of a nucleicacid encoding a MAP protein, and/or modifying in a plant level and/oractivity of a MAP protein, gives plants having modified growthcharacteristics.

Accordingly, the present invention provides a method for modifying plantgrowth characteristics relative to corresponding wild type plants,comprising modifying expression in a plant of a nucleic acid encoding amethionine aminopeptidase (MAP) protein and/or modifying level and/oractivity in a plant of a MAP protein.

The term “modifying” as used herein is taken to mean increasing,decreasing and/or changing in place and/or time. Modifying expression ofa nucleic acid encoding a MAP protein or modifying level and/or theactivity of a MAP protein encompasses altered expression of a geneand/or altered level and/or activity of a gene product namely apolypeptide, in specific cells or tissues, when compared to expression,level and/or activity of a MAP gene or protein in correspondingwild-type plants. The modified gene expression may result from modifiedexpression of an endogenous MAP gene and/or may result from modifiedexpression of a MAP gene previously introduced into a plant. Similarly,modified levels and/or activity of a MAP protein may be due to modifiedexpression of an endogenous MAP nucleic acid or protein and/or due tomodified expression of a MAP nucleic acid or protein previouslyintroduced into a plant. Modified expression of a nucleic acid/geneand/or modified level and/or activity of a gene product/protein may beeffected, for example, by chemical means and/or recombinant means.

According to a preferred embodiment of the present invention, modifyingexpression of a nucleic acid encoding a MAP protein and/or modifyinglevel and/or activity of a MAP protein may be effected by recombinantmeans. Such recombinant means may comprise a direct and/or indirectapproach.

For example, an indirect recombinant approach may comprise introducing,into a plant, a nucleic acid capable of modifying expression of a MAPgene and or capable of modifying level and/or activity of a MAP protein.Examples of such nucleic acids to be introduced into a plant includenucleic acids encoding transcription factors or activators or inhibitorsthat bind to the promoter of a MAP gene or that interact with a MAPprotein. Methods to test these types of interactions and methods forisolating nucleic adds encoding such interactors include yeastone-hybrid or yeast two-hybrid screens in which the MAP gene/protein isused as bait. Therefore, modifying expression of a nucleic acid encodinga MAP protein and/or modifying level and/or activity of the MAP proteinmay be effected by decreased or increased levels of factors that controlexpression of the MAP gene or that directly or indirectly (in)activatethe MAP protein. Further, modifying level and/or activity of a MAPprotein may be effected by modifying levels of a factor capable ofinteracting with a MAP protein. Such factors may include a ligand or anatural target/substrate of a MAP protein. An example of such a targetincludes the eukaryotic initiation factor eIF2, which is part of theprotein translation initiation complex.

Also encompassed by an indirect recombinant approach for modifyingexpression of a MAP gene and/or modifying level and/or activity of a MAPprotein is the provision of, or the inhibition or stimulation ofregulatory elements that drive expression of MAP gene, for example theendogenous MAP gene. For example, such regulatory elements to beintroduced into a plant may be a promoter capable of driving expressionof an endogenous MAP gene.

A preferred recombinant approach for modifying expression of a nucleicacid encoding a MAP protein and/or modification of level and/or activityof a MAP protein comprises introducing into a plant a nucleic acidcapable of modifying expression of a nucleic acid encoding a MAP proteinand/or capable of modifying level and/or activity of a MAP protein.

Accordingly, the present invention provides a method for modifying plantgrowth characteristics as described above, wherein the modifyingexpression, level and/or activity is effected by introducing into aplant a nucleic acid capable of modifying expression of a nucleic acidencoding a MAP protein and/or capable of modifying level and/or activityof a MAP protein. According to a more direct and further preferredembodiment of such a method, that nucleic acid is a nucleic acidencoding a MAP protein or a variant thereof as described herein below,or is a variant of a nucleic acid encoding a MAP protein. This nucleicacid encoding a MAP protein may be wild type, i.e. the native orendogenous. Alternatively, the nucleic acid may be heterologous, i.e.derived from the same or another species, which nucleic acid isintroduced as a transgene. This transgene may be substantially modifiedfrom its native form in composition and/or genomic environment throughdeliberate human manipulation.

Additionally or alternatively, modifying expression of the nucleic acidencoding a MAP protein and/or modifying level and/or activity of the MAPprotein itself may be effected by a chemical approach. Such a chemicalapproach may involve exogenous application of one or more compounds orelements capable of modifying expression of a MAP nucleic acid(endogenous gene or introduced into the plant) and/or capable ofmodifying level and/or activity of a MAP protein (endogenous orintroduced into the plant). The term “exogenous application” as definedherein is taken to mean the contacting or administering of a suitablecompound or element to a plant, plant cell, tissue or organ. Thecompound or element may be exogenously applied to a plant in a formsuitable for plant uptake (such as through application to the soil foruptake via the roots, or in the case of some plants, by applyingdirectly to the leaves, for example by spraying). The exogenousapplication may take place on wild-type plants or on transgenic plantsthat have previously been transformed with a MAP nucleic acid/gene orother transgene.

Suitable compounds or elements for exogenous application include MAPproteins or MAP nucleic acids. Alternatively, suitable compounds orelements include those capable of directly or indirectly binding or(in)activating a MAP protein. Suitable compounds also include antibodiesthat can recognise or mimic the function of a MAP protein. Suchantibodies may comprise “plantibodies”, single chain antibodies, IgGantibodies and heavy chain camel antibodies, as well as fragmentsthereof. Other suitable compounds or elements for chemical modificationof expression, activity and/or level of a MAP gene or protein, includemutagenic substances, such as N-nitroso-N-ethylurea, ethylene imine,ethyl methanesulphonate and diethyl sulphate. Mutagenesis may also beachieved by exposure to ionising radiation, such as X-rays or gamma-raysor ultraviolet light. Methods for introducing mutations, and for testingthe effect of mutations (such as by monitoring gene expression and/orprotein activity), are well known in the art.

Therefore, according to one aspect of the present invention, there isprovided a method for modifying plant growth characteristics, comprisingexogenous application of one or more compounds or elements capable ofmodifying expression of a MAP gone and/or capable of modifying leveland/or activity of a MAP protein.

Methionine aminopeptidases (MetAP or MAP) are described in literature tobe responsible for removal of the initiator methionine residue of apeptide during protein synthesis (Bradshaw and Yi, 2002, Essays Biochem38:65-78). Methionine aminopeptidases are specific and ubiquitousenzymes that belong to the family of metalloenzymes. It has been foundthat Eukaryotes have two classes of methionine aminopeptidase (MAP1 andMAP2), while prokaryotes only have one. MAP2 is also known as theeukaryotic initiation factor 2 alpha (eIF2alpha) associated protein p67.It has been demonstrated that rat p67 In addition to its peptidasefunction, also plays an important role in translational regulation bypreventing the phosphorylation of the alpha subunit of initiationfactor-2. Accordingly, MAP2 proteins have, in addition to theirpeptidase activity, a non-proteolytic function to protect eIF2alphaagainst phosphorylation (POEP), which eIF2alpha is inactive in thephosphorylated state (Datta R et al. Biochimie. 2001 83:919-31). MAPproteins from various organisms have been studied with respect to theirfunction as well as their structure. Based on sequence analysis, MAPproteins have been found to have a MAP signature and a peptidase domain.For example, typical structural features of a MAP1 protein are a MAP1signature (PROSITEPS00680=[MFY]-x-G-H-G-[LIVMC]-[GSH]-x(3pH-x(4)-[LIVM]-x-[HN]-[YWVH]) anda pFAM PEPTIDASE_M24 domain. Typical structural features of a MAP2protein include a MAP2 signature (PROSITEPS01202=[DA]-[LIVMY]-x-K-[LIVM]-D-x-G-x-[HQ]-[LIVM]-[DNS]-G-x(3)-[DN])and a pFAM PEPTIDASE_M24 domain. MAP2 proteins, such as plant MAP2proteins, additionally comprise at least one lysine-rich domain at theN-terminus. MAP proteins isolated from yeast have been described in Liand Chang, (1995) Proc Natl Acad Sci. USA. 92(26): 12357-61, Human MAPproteins have been described in Li and Chang (1996) Biochem Biophys ResCommun 227:152-159 and six MAP cDNAs were cloned from Arabidopsisthaliana and corresponding proteins were characterized in vivo and invitro (Giglione et al. EMBO J. 2000 19:5916-29). One example of anArabidopsis thaliana MAP protein is herein represented by SEQ ID NO 2,and its encoding sequence by SEQ ID NO 1. Another example of anArabidopsis thaliana MAP protein is herein represented by SEQ ID NO 4,and its encoding sequence by SEQ ID NO 3.

The term “MAP protein” as used herein encompasses a methionineaminopeptidases (MAP), for example a MAP of SEQ ID NO 2 or 4, as well asa variant thereof (or a proteins essentially similar thereto). The terms“MAP gene” or “MAP nucleic acid” or “nucleic acid encoding a MAPprotein” are used herein interchangeably and also encompass variant MAPnucleic acids, for example variants of SEQ ID NO 1 or 3. The terms “avariant of” and “essentially similar to” are used interchangeablyherein. Variant MAP proteins or variant nucleic acids encoding a MAPprotein include:

-   -   (i) Functional portions of a MAP nucleic acid, for example a MAP        nucleic acid of SEQ ID NO 1 or 3;    -   (ii) Sequences capable of hybridising with a MAP nucleic acid,        for example with a MAP nucleic acid of SEQ ID NO 1 or 3;    -   (iii) Alternative splice variants of a MAP nucleic acid, for        example a MAP nucleic acid of SEQ ID NO 1 or 3;    -   (iv) Allelic variants of a MAP nucleic acid, for example a MAP        nucleic acid of SEQ ID NO 1 or 3; and    -   (v) Homologues, derivatives and active fragments of a MAP        protein, for example a MAP protein of SEQ ID NO 2 or 4.

Advantageously, the methods according to the invention may be practisedusing variant MAP proteins and variant MAP nucleic acids. Suitablevariants include variants of SEQ ID NO 2 or 4 and/or variants of SEQ IDNO 1 or 3. However, it should be clear that the applicability of theinvention is not limited to the use of the nucleic acid represented bySEQ ID NO 1 or 3, nor to the nucleic acid encoding the amino acidsequence represented by SEQ ID NO 2 or 4, but that other nucleic acidsencoding variants of SEQ ID NO 2 may be useful in the methods of thepresent invention.

For use in the methods according to the present invention, the MAPprotein preferably comprises any one or both of the following domains:

-   -   a) a MAP signature    -   b) a peptidase_M24 domain

A preferred MAP protein comprises both of these domains in the aboverank order.

The term “variant” also includes variants in the form of a complement,DNA, RNA, cDNA or genomic DNA. The variant nucleic acid may besynthesized in whole or in part, it may be a double-stranded nucleicacid or a single-stranded nucleic acid. Also, the term “variant”encompasses a variant due to the degeneracy of the genetic code; afamily member of the gene or protein; and variants that are interruptedby one or more intervening sequences such as introns or transposons.

One variant nucleic acid encoding a MAP protein is a functional portionof a nucleic acid encoding a MAP protein. Advantageously, the method ofthe present invention may also be practised using portions of a nucleicacid encoding a MAP protein. A functional portion refers to a piece ofDNA derived from or prepared from an original (larger) DNA molecule,which portion, retains at least part of the functionality of theoriginal, DNA and which, when expressed in a plant, gives plants havingmodified growth characteristics. The portion may comprise many genes,with or without additional control elements or may contain spacersequences. The portion may be made by making one or more deletionsand/or truncations to the nucleic acid. Techniques for introducingtruncations and deletions into a nucleic acid are well known in the art.Portions suitable for use in the methods according to the invention mayreadily be determined by following the methods described in the Examplessection by simply substituting the sequence used in the actual Examplewith the portion to be tested for functionality.

Another variant of a nucleic acid encoding a MAP protein is a nucleicacid capable of hybridising with a nucleic acid encoding a MAP protein,for example to any of the nucleic acids encoding a protein representedby SEQ ID NO 2 or 4. Hybridising sequences suitable for use in themethods according to the invention may readily be determined, forexample by following the methods described in the Examples section bysimply substituting the sequence used in the actual Example with thehybridising sequence.

The term “hybridising” as used herein means annealing to a substantiallyhomologous complementary nucleotide sequences in a hybridizationprocess. The hybridisation process can occur entirely in solution, i.e.both complementary nucleic acids are in solution. Tools in molecularbiology relying on such a process include the polymerase chain reaction(PCR; and all methods based thereon), subtractive hybridisation, randomprimer extension, nuclease S1 mapping, primer extension, reversetranscription, cDNA synthesis, differential display of RNAs, and DNAsequence determination. The hybridisation process can also occur withone of the complementary nucleic acids immobilised to a matrix such asmagnetic beads, Sepharose beads or any other resin. Tools in molecularbiology relying on such a process include the isolation of poly (A+)mRNA. The hybridisation process can furthermore occur with one of thecomplementary nucleic acids immobilised to a solid support such as anitro-cellulose or nylon membrane or immobilised by e.g.photolithography to e.g. a siliceous glass support (the latter known asnucleic acid arrays or microarrays or as nucleic acid chips). Tools inmolecular biology relying on such a process include RNA and DNA gel blotanalysis, colony hybridisation, plaque hybridisation, in situhybridisation and microarray hybridisation. In order to allowhybridisation to occur, the nucleic acid molecules are generallythermally or chemically denatured to melt a double strand into twosingle strands and/or to remove hairpins or other secondary structuresfrom single stranded nucleic acids. The stringency of hybridisation isinfluenced by conditions such as temperature, sodium/salt concentrationand hybridisation buffer composition. High stringency conditions forhybridisation include high temperature and/or low salt concentration(salts include NaCl and Na₃-citrate) and/or the inclusion of formamidein the hybridisation buffer and/or lowering the concentration ofcompounds such as SDS (sodium dodecyl sulphate detergent) in thehybridisation buffer and/or exclusion of compounds such as dextransulphate or polyethylene glycol (promoting molecular crowding) from thehybridisation buffer. Conventional hybridisation conditions aredescribed in, for example, Sambrook (2001) Molecular Cloning: alaboratory manual, 3rd Edition Cold Spring Harbor Laboratory Press, CSH,New York, but the skilled craftsman will appreciate that numerousdifferent hybridisation conditions can be designed in function of theknown or the expected homology and/or length of the nucleic acid.Sufficiently low stringency hybridisation conditions are particularlypreferred (at least in the first instance) to isolate nucleic acidsheterologous to the DNA sequences of the invention defined supra. Anexample of low stringency conditions is 4-6×SSC/0.1-0.5% w/v SDS at37-45° C. for 2-3 hours. Depending on the source and concentration ofthe nucleic acid involved in the hybridisation, alternative conditionsof stringency may be employed, such as medium stringency conditions.Examples of medium stringency conditions include 1-4×SSC/0.25% w/v SDSat ≧45° C. for 2-3 hours. With specifically hybridising is meanthybridising under stringent conditions. An example of high stringencyconditions includes 0.1-2×SSC, 0.1×SDS, and 1×SSC, 0.1×SDS at 60° C. for2-3 hours.

The methods according to the present invention may also be practisedusing an alternative splice variant of a nucleic acid encoding a MAPprotein, for example an alternative splice variant of SEQ ID NO 1 or 3.The term “alternative splice variant” as used herein encompassesvariants of a nucleic acid in which selected introns and/or exons havebeen excised, replaced or added. Such splice variants may be found innature or may be manmade. Methods for making such splice variants arewell known in the art. Splice variants suitable for use in the methodsaccording to the invention may readily be determined, for example, byfollowing the methods described in the Examples section by simplysubstituting the sequence used in the actual Example with the splicevariant.

Another variant MAP nucleic acid useful in practising the method formodifying plant growth characteristics, is an allelic variant of a MAPgene, for example an allelic variant of SEQ ID NO 1 or 3. Allelicvariants exist in nature and encompassed within the methods of thepresent invention is the use of these natural alleles. Allelic variantsalso encompass Single Nucleotide Polymorphisms (SNPs) as well as SmallInsertion/Deletion Polymorphisms (INDELs). The size of INDELs is usuallyless than 100 bp. SNPs and INDELs form the largest set of sequencevariants in naturally occurring polymorphic strains of most organisms.Allelic variants suitable for use in the methods according to theinvention may readily be determined for example by following the methodsdescribed in the Examples section by simply substituting the sequenceused in the actual Example with the allelic variant.

The present invention provides a method for modifying plant growthcharacteristics, comprising modifying expression in a plant of analternative splice variant or of an allelic variant of a nucleic acidencoding a MAP protein and/or by modifying level and/or activity in aplant of a MAP protein encoded by an alternative splice variant orallelic variant.

One example of a variant MAP protein useful in practising the methods ofthe present invention is a homologue of a MAP protein. “Homologues” of aparticular MAP protein encompass peptides, oligopeptides, polypeptides,proteins and enzymes having an amino acid substitution, deletion and/orinsertion relative to that particular MAP protein and having similarbiological and functional activity as a MAP protein. Homologues of a MAPproteins may be manmade via the techniques of genetic engineering and/orprotein engineering. To produce such homologues, amino acids of theprotein may be replaced by other amino acids having similar properties(such as similar hydrophobicity, hydrophilicity, antigenicity,propensity to form or break α-helical structures or β-sheet structures).Conservative substitution tables are well known in the art (see forexample Creighton (1984) Proteins. W.H. Freeman and Company).

Additionally and/or alternatively, homologues of a particular MAPprotein exist in nature and may be found in the same or differentspecies or organism from which the particular MAP protein is derived.Two special forms of homologues, orthologues and paralogues, areevolutionary concepts used to describe ancestral relationships of genes.The term “orthologues” relates to homologous genes in differentorganisms due to ancestral relationship. The term “paralogues” relatesto gene-duplications within the genome of a species leading toparalogous genes. The term “homologues” of a MAP protein as used hereintherefore encompasses paralogues and orthologues of the MAP protein,which are also useful for practising the methods of the presentinvention.

The homologues useful in the method according to the invention have, inincreasing order of preference, at least 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 58%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% sequence identity with the sequence of SEQ ID NO 2or with the sequence of SEQ ID NO 4, preferably over the full length ofSEQ ID NO 2 or 4.

The percentage of sequence identity is calculated using a pairwiseglobal alignment program implementing the algorithm of Needleman-Wunsch(J. Mol. Biol. 48: 443-453, 1970) which maximizes the number of matchesand minimizes the number of gaps. For calculation of the above-mentionedpercentages, the program Align X (as part of the Vector NTI suite 5.5)is used with the standard parameters and the variable parameters gapopening penalty 10 and gap extension penalty 0.1.

Multiple alignment of MAP proteins from various sources is generatedusing the program Clustal X with the standard fixed parameters and thevariable parameters Gap opening penalty 10 and Gap extension penalty0.2. Percentage of identity between the proteins of this multiplealignment was calculated using the BoxShade software (available athttp://www.isrec.isb-sib.ch/ftp-server/boxshade/). Table I gives anoverview of these percentages. TABLE I Sequence identity betweendifferent MAP proteins General % identity Sequences used for calculationBetween MAP2 of dicots 90% 2 Arabidopsis sequences Between MAP2 ofmonocots 80% 2 rice and 1 maize sequences Between MAP2 of dicots andmonocots 76% 3 monocot and 2 dicot sequences Between MAP2 of plants andanimals 65% 5 plant and 4 animal sequences Between MAP1 and MAP2. 20% 11MAP2 and 6 MAP1 sequences

The homologues useful in the methods according to the invention may bederived (either directly or indirectly, i.e. if subsequently modified)from any source provided that the sequence, when expressed in a plant,leads to modified growth characteristics. The nucleic acid may beisolated from a microbial source, such as bacteria, yeast or fungi, orfrom a plant, algae, insect or animal (including human) source.

The nucleic acid encoding a MAP homologue is preferably isolated from aplant. Further preferably, the nucleic acid is isolated from adicotyledoneous plant, preferably from the family Bressicaceae, furtherpreferably from Arabidopsis thaliana. MAP proteins of Arabidopsis havebeen subdivided into different classes on the basis of their level ofsequence homology (Giglione et al. (2000, EMBO J. 19: p 5916-5929).Class MAP1 (with A, B, C and D isoforms) and class MAP2 (with A and Bisoforms) were identified in Arabidopsis. These classes and isoforms arealso encompassed by the term “homologue” as used herein. Advantageously,these different classes or isoforms of MAP proteins, or their encodingnucleic acids, may be used in the methods of the present invention.Accordingly, the present invention provides a method as describedhereinabove, wherein the MAP nucleic acid or MAP protein is obtainedfrom a plant, preferably from a dicotyledoneous plant, furtherpreferably from the family Brassicaceae, more preferably fromArabidopsis thaliana. According to a further embodiment, a MAP nucleicacid or protein is a MAP2 nucleic acid or protein or a MAP1 nucleic acidor protein. According to a further embodiment of the invention, the MAPnucleic acid encodes or the MAP protein is, an isoform of the MAPprotein represented by SEQ ID NO 2 or 4. According to a furtherembodiment of the invention, the MAP nucleic acid encodes or the MAPprotein is the Arabidopsis thaliana MAP2B protein represented by SEQ IDNO 2.

Other preferred MAP homologues and their encoding sequences may be foundin (public) sequence databases. Methods for the search andidentification of MAP homologues in sequence databases would be wellwithin the realm of persons skilled in the art. Such methods, involvescreening sequence databases with the sequences provided by the presentinvention, for example SEQ ID NO 2 or 4 (or SEQ ID NO 1 or 3),preferably in a computer readable form. Useful sequence databasesinclude, but are not limited, to Genbank(http://www.ncbi.nim.nih.gov/web/Genbank), the European MolecularBiology Laboratory Nucleic acid Database (EMBL)(http://w.ebi.ac.uk/ebi-docs/embl-db.html) or versions thereof or theMIPS database (http://mips.gsf.del). Different search algorithms andsoftware for the alignment and comparison of sequences are well known inthe art. Such software includes for example GAP, BESTFIT, BLAST, FASTAand TFASTA. Preferably the BLAST software is used, which calculatespercent sequence identity and performs a statistical analysis of thesimilarity between the sequences. The suite of programs referred to asBLAST programs has 5 different implementations: three designed fornucleotide sequence queries (BLASTN, BLASTX, and TBLASTX) and twodesigned for protein sequence queries (BLASTP and TBLASTN) (Coulson,Trends in Biotechnology: 76-80, 1994; Birren et al., GenomeAnalysis, 1:543, 1997). The software for performing BLAST analysis is publiclyavailable through the National Centre for Biotechnology Information.

Orthologues of a MAP protein in other plant species may easily be foundby performing a reciprocal Blast search. This method encompassessearching one or more sequence databases with a query gene or protein ofinterest (for example SEQ ID NO 1, 2, 3 or 4), using for example theBLAST program. The highest-ranking subject genes that result from thesearch are then used as a query sequence in a similar BLAST search. Onlythose genes that have as a highest match again the original querysequence (SEQ ID NO 1, 2, 3 or 4) are considered to be orthologousgenes. For example, to find a rice orthologue of an Arabidopsis thalianagene, one may perform a BLASTN or TBLASTX analysis on a rice databasesuch as (but not limited to) the Oryza sativa Nipponbare databaseavailable at the NCBI website (http://wwww.ncbi.nlm.nih.gov) or thegenomic sequences of rice (cultivars indica or japonica). In a nextstep, the obtained rice sequences are used in a reverse BLAST analysisusing an Arabidopsis database. The method can be used to identifyorthologues from many different species, for example from corn.

Paralogues of a MAP protein in the same species may easily be found byperforming a Blast search on sequences of the same species from whichthe MAP protein is derived. From the sequences that are selected by theBlast search, the true paralogues may be identified by looking foridentity between the sequences or for conservation of typical MAPdomains. A search for paralogues of the Arabidopsis thaliana MAP proteinhas been carried out by Giglione et al. (2000, EMBO J. 19: p 5916-5929).

A BLAST using default parameters and using SEQ ID NO 2 or SEQ ID NO 1 assearch sequence was performed and resulted in the identification of thefollowing MAP nucleic acids and proteins. Sequences substantiallyidentical to SEQ ID NO 1 (AtMAP2B) have been published under Genbankaccession numbers NM-115862 (genomic DNA), BT000063 (At3g59990 mRNA),AY084710, AY065161 and AF300B80. One isoform of SEQ ID NO 1 and 2, namedherein AtMAP2A, was found in the database under Genbank accession numberAF250964. Isoforms of a different dass were found in the database underGenbank accession numbers AF250960 ((AtMAP1A and represented herein bySEQ ID NO 3 and SEQ ID NO 4), AF250961 (AtMAP1B), AF250962 (AtMAP1C) andAF250963 (AtMAP1D).

Also MAP homologues from different plant species have been identified,which homologues are useful in the methods of the present invention.There is a high degree of conservation among the MAP proteins of plants(see Table I). Such homologues include for example, the MAP protein fromOryza sativa as published in Genbank database under accession numberBAD03108, the Oryza sativa protein under accession number AK122063, theOryza sativa protein under Genbank accession number AK107616 and the ZeeMays protein under Genbank accession number AY105027. The genes encodingMAP homologues of crop plants may be especially useful in practising themethods of the invention in crop plants. In another embodiment of theinvention, the genes encoding MAP homologues of a dicot plant may beused to practise the methods of the present invention in a monocotplant, or vice versa.

The genome sequences of Arabidopsis thaliana and Oryza sativa are nowavailable in public databases such as Genbank and other genomes arecurrently being sequenced. Therefore, it is expected that furtherhomologues will readily be identifiable by sequence alignment with SEQID NO 1 or 3 or with SEQ ID NO 2 or 4 using the programs Blast X orBlastP or other programs.

A phylogenetic tree may be constructed with all the homologues,paralogues and orthologues as defined hereinabove. Multiple alignmentsare made using the program ClustalX as described hereinabove. Thephylogenetic tree is made by the Phylip software package available athttp://evolution.genetics.washington.edu/phylip.html. Sequencesclustering around one or more of the 6 MAP proteins of Arabidopsisthaliana identify proteins, and their corresponding genes, suitable foruse in the methods of the present invention.

The above-mentioned software analyses for comparing sequences, for thecalculation of sequence identity, for the search for homologues,orthologues or paralogues or for the making of a phylogenetic tree, ispreferentially done with full-length sequences. Alternatively, thesesoftware analyses may be carried out on with a conserved region of theMAP protein or DNA sequence. Accordingly, these analyses may be based onthe comparison and calculation of sequence identity between conservedregions, functional domains, motifs or boxes.

The identification of such domains or motifs, for example, the domainsrepresented by SEQ ID NO 5 (MAP1 signature), SEQ ID NO 6 (MAP2signature), SEQ ID NO 7 (peptidase-M24 domain of AtMAP1A), SEQ ID NO 8(peptidase-M24 domain of AtMAP2B), SEQ ID NO 9 (lysine-rich domain ofAtMAP2B), would also be well within the realm of a person skilled in theart and involves screening a computer readable format of MAP proteinsfor the presence of conserved protein domains, motifs and boxes. Proteindomain information is available in the PRODOM(http://www.biochem.ucl.ac.uk/bsm/dbbrowser/jj/prodomsrchjj.html), PIR(http://pir.georgetown.edu/), PROSITE (http://au.expasy.org/PROSITE/) orpFAM (http://pFAM.wustl.edu/) database. Software programs designed forsuch domain searching include, but are not limited to, MotifScan, MEME,SIGNALSCAN, and GENESCAN. MotifScan is a preferred software program andis available at (http://hits.isb-sib.ch/cqi-bin/PFSCAN, which programuses the protein domain information of PROSITE and pFAM. A MEMEalgorithm (Version 3.0) may be found in the GCG package; or athttp://www.sdsc.edu/MEME/meme. SIGNALSCAN version 4.0 information isavailable at http://biosci.cbs.umn.edu/software/sigscan.html. GENESCANmay be found at http://gnomic.stanfbrd.edu/GENESCANW.html.

The term “MAP signature” means a MAP-specific domain. Examples of suchMAP signatures are MAP1 or MAP2 signatures. The MAP1 signature isdescribed in the PROSITE database under accession number PS00680 and isherein represented by the consensus sequence of SEQ ID NO 5:[MFY]-x-G-H-G[LIVMC]-[GSH]-x(3)-H-x(4)-[LIVM]-x-[HN]-[YWVH]. In MAP1proteins, the MAP1 signature may be located within the peptidase domain.The MAP2 signature is further described in the PROSITE database underaccession number PS01202 and is herein represented by the consensussequences of SEQ ID NO 6:[DA]-[LIVMY]-x-K-[LIVM]-D-x-G-x-[HQ]-[LIVM]-[DNS]-G-x(3)-[DN]. In caseof MAP2 proteins, the MAP2 signature is preferably located upstream ofthe peptidase domain. A person skilled in the art will recognize that aMAP signature may deviate, with for example 1 or 2 mismatches, from theabove-mentioned consensus MAP signatures, without loosing itsfunctionality. One example is found in the Drosophila MAP2 protein (seeFIG. 3), having a K at the twelfth position of its MAP2 signature,instead of D, N, or S.

The term “peptidase_M24 domain”, as used herein, refers to a peptidasedomain, which occurs in MAP proteins. The peptidase-M24 domain isdescribed in the pFAM database under accession number PF00557. Aconsensus sequence for this domain is not given in the PFAM database,but nearly 500 proteins were categorized as having a PEPTIDASE_M24domain. This domain may be identified by its folding and tertiarystructure, rather than by its primary structure, which may be variable.Different MAP proteins therefore also exhibit substantial variation inthe primary structure (amino acid sequence) of this peptidase_M24 domain(see FIG. 3). A person skilled in the art would readily know how todetermine the presence of a peptidase_M24 domain in a protein sequence.One example is to submit the protein sequence to a software programcapable of determining conserved domains, for example the programMotifScan as described hereinbefore. One example of a peptidase_M24domain is given in SEQ ID NO 7, which is the peptidase_M24 domain ofAtMAP1A. Another example is given in SEQ ID NO 8, which is thepeptidase-M24 domain of AtMAP2B. Preferably, the MAP protein used in themethods of the present invention has a peptidase_M24 domain, which is atleast 70% identical to SEQ ID NO 7 or to SEQ ID NO 8.

Optionally, for example the case of MAP2 proteins, the MAP proteinuseful in the methods of the present invention has at least onelysine-rich domain. Preferably such lysine-rich domain is locatedbetween the N-terminus and the MAP signature. The term “lysine-richregion” means an amino acid region, which is enriched with lysine aminoacids. Typically a lysine-rich domain is an amino acid sequence of whichmore than 50% of the amino acids are lysine (K). For example, inAtMAP2B, 12 out of 14 continuous residues of the lysine-rich domain arelysine, which corresponds to 85% of lysine residues. Optionally astretch of continuous lysines may be present for example a stretch of atleast 3 lysine residues, for example of 4, 5, 6 or more lysine residues.Accordingly, substantial variation between the lysine-rich domains ofdifferent MAP proteins exists, as is illustrated in FIG. 3. Thelysine-rich domain of the Arabidopsis thaliana MAP2B protein isrepresented by SEQ ID NO 9.

Based on the presence and conservation of the above-mentioned structuraldomains, persons skilled in the art have been able to readily recognizeMAP proteins of different organism, for example from plants (seeGiglione et al., (2000), EMBO J. 19: p 5916-5929).

Some of the variants as mentioned hereinabove may occur in nature. Oncethe sequence of a variant is known, and its corresponding codingsequence, the person skilled in the art will be able to isolate thecorresponding MAP gene or variant from biological material, for exampleby the technique of PCR. One example of such an experiment is outlinedin Example 1.

Alternatively and/or additionally, the variants as mentioned above maybe manmade via techniques involving, for example, mutation(substitution, insertion or deletion) or derivation. These variants areherein referred to as “derivatives”, which derivatives are also usefulin the methods of the present invention. Derivatives of a protein mayreadily be made using peptide synthesis techniques well known in theart, such as solid phase peptide synthesis and the like, or byrecombinant DNA manipulations. The manipulation of DNA sequences toproduce substitution, insertion or deletion variants of a protein arewell known in the art. For example, techniques for making substitutionmutations at predetermined sites in DNA are well known to those skilledin the art and include M13 mutagenesis, T7-Gen in vitro mutagenesis(USB, Cleveland, Ohio), QuickChange Site Directed mutagenesis(Stratagene, San Diego, Calif.), PCR-mediated site-directed mutagenesisor other site-directed mutagenesis protocols.

One example of a derivative is a substitutional variant. The term“substitutional variants” of a protein refers to those variants in whichat least one residue in an amino acid sequence has been removed and adifferent amino acid inserted in its place. Amino acid substitutions aretypically of single residues, but may be clustered depending uponfunctional constraints placed upon the polypeptide; insertions usuallyare of the order of about 1-10 amino adds, and deletions can range fromabout 1-20 amino acids. Preferably, amino add substitutions compriseconservative amino add substitutions.

Other derivatives are “insertional variants” in which one or more aminoacids are introduced into a predetermined site in the MAP protein.Insertions may comprise amino-terminal and/or carboxy-terminal fusion aswell as intra-sequence insertion of single or multiple amino acids.Generally, insertions within the amino acid sequence will are of theorder of about 1 to 10 amino acids. Examples of amino- orcarboxy-terminal fusions include fusion of the binding domain oractivation domain of a transcriptional activator as used in the yeasttwo-hybrid system, phage coat proteins, (histidine)₆-tag, glutathioneS-transferase-tag, protein A, maltose-binding protein, dihydrofolatereductase, Tag•100 epitope, c-myc epitope, FLAG®-epitope, lacZ, CMP(calmodulin-binding peptide), HA epitope, protein C epitope and VSVepitope.

Other derivatives of a MAP protein are “deletion variants”,characterised by the removal of one or more amino acids from theprotein.

Another “derivative” of a MAP protein is characterised by substitutions,and/or deletions and/or additions of naturally and non-naturallyoccurring amino acids compared to the amino acids of anaturally-occurring MAP protein. A derivative may also comprise one ormore non-amino acid substituents compared to the amino acid sequencefrom which it is derived. Such non-amino acid substituents include forexample non-naturally occurring amino acids, a reporter molecule orother ligand, covalently or non-covalently bound to the amino acidsequence. Such as reporter molecule may be bound to facilitate thedetection of the MAP protein.

Another variant of a MAP protein useful in the methods of the presentinvention is an active fragment of a MAP protein. “Active fragments” ofa MAP protein encompass at least five contiguous amino acid residues ofa MAP protein, which residues retain similar biological and/orfunctional activity to a naturally occurring protein or a part thereof.Suitable fragments include fragments of a MAP protein starting at thesecond or third or further internal methionine residues. These fragmentsoriginate from protein translation, starting at internal ATG codons.Functional fragments of a MAP protein useful in practising the methodsof the present invention may have one or more of the conserved domainsof a MAP proteins, whilst retaining its functionality in the methods ofthe present invention.

According to a preferred embodiment of the present invention, a methodfor modifying plant growth characteristics, comprises enhanced orincreased expression of a nucleic acid encoding a MAP protein. Methodsfor obtaining enhanced or increased expression of genes or gene productsare well documented in the art and include, for example, overexpressiondriven by a (strong) promoter, the use of transcription enhancers ortranslation enhancers. The term overexpression as used herein means anyform of expression that is additional to the original wild-typeexpression level. Preferably the nucleic acid to be introduced into theplant and/or the nucleic acid that is to be overexpressed in the plantis in the sense direction with respect to the promoter to which it isoperably linked. Preferably, in the methods of the present invention anucleic acid encoding a MAP protein is overexpressed in a plant, such asa MAP nucleic acid of SEQ ID NO 1 or a variant thereof, such as aportion of SEQ ID NO 1 or a sequence capable of hybridising therewith.However, it should be clear that the applicability of the invention isnot limited to use of the nucleic acid represented by SEQ ID NO 1 nor tothe nucleic acid encoding the amino acid sequence of SEQ ID NO 2, butthat other nucleic acids encoding homologues, derivatives or activefragments of SED ID NO 2 may be useful in the methods of the presentinvention.

Alternatively and/or additionally, increased expression of a MAP gene orincreased level, and/or activity of a MAP protein in a plant cell, isachieved by mutagenesis. For example these mutations may be responsiblefor altered control of the MAP gene, resulting in more expression of thegene, relative to the wild-type gene. Mutations can also causeconformational changes in a protein, resulting in higher levels and/ormore activity of the MAP protein. Such mutations or such mutant genesmay be selected, or isolated and/or introduced into the same ordifferent plant species in order to obtain plants having modified growthcharacteristics. Examples of such mutants include dominant positivemutants of a MAP gene.

Modifying gene expression (whether by a direct or indirect approach)encompasses altered transcript levels of a gene. Altered transcriptlevels can be sufficient to induce certain phenotypic effects, forexample via the mechanism of co-suppression. Here the overall effect ofoverexpression of a transgene is that there is less activity in the cellof the protein encoded by a native gene having homology to theintroduced transgene. Therefore, according to another embodiment of thepresent invention, there is provided a method for modifying plant growthcharacteristics, comprising inhibiting or decreasing expression of agene encoding a MAP protein or decreasing level and/or activity of a MAPprotein. Examples of decreasing expression, level and/or activity of aprotein are also well documented in the art and include, for example,downregulation of expression by anti-sense techniques, RNAi techniques,small interference RNAs (siRNAs) and microRNA (mRNA).

Another method for downregulation of gene expression or gene silencingcomprises use of ribozymes, for example as described in Atkins et al.1994 (WO 94/00012), Lenee et al. 1995 (WO 95/03404), Lutziger et al.2000 (WO 00/00619), Prinsen et al. 1997 (WO 97/3865) and Scott et al.1997 (WO 97/38116).

Gene silencing may also be achieved by insertion mutagenesis (forexample, T-DNA insertion or transposon insertion) or by gene silencingstrategies as described by, among others, Angell and Baulcombe 1998 (WO98/36083), Lowe et al. 1989 (WO 98/53083), Lederer et al. 1999 (WO99/15682) or Wang et al. 1999 (WO 99/53050).

Expression of an endogenous MAP gene may also be reduced by mutation.Such a mutation or such a mutant gene may be isolated and introducedinto the same or different plant species in order to obtain plantshaving modified growth characteristics. Examples of such mutants includedominant negative mutants of a MAP gene.

Genetic constructs aimed at silencing gene expression may comprise thenucleotide sequence encoding a MAP protein, for example a nucleic acidrepresented by SEQ ID NO 1 (or a variant thereof), in a sense and/oranti-sense orientation relative to the promoter sequence. The senseand/or anti-sense copies of at least part of the endogenous gene in theform of direct or inverted repeats or in the form of a hairpin may beutilised in the methods according to the invention. The growth of plantsmay also be modified by introducing into a plant at least part of ananti-sense version of a nucleotide sequence encoding a MAP protein.

According to a further aspect of the present invention, there isprovided genetic constructs and vectors to facilitate introduction intoa plant cell and/or facilitate expression and/or facilitate maintenanceof the nucleotide sequence capable of modifying expression of a nucleicacid encoding a MAP protein and/or capable of modifying level and/oractivity of a MAP protein. Therefore, according to a further aspect ofthe present invention, there is provided a genetic construct comprising:

-   -   (a) A nucleic acid encoding a plant MAP protein or a variant        thereof or a variant of a nucleic acid encoding a plant MAP        protein;    -   (b) One or more control sequences capable of driving expression        of the nucleic acid of in a plant (a); and optionally    -   (c) A transcription termination sequence.

Constructs useful in the methods according to the present invention maybe constructed using recombinant DNA technology well known to personsskilled in the art. The genetic constructs may be inserted into vectors,which may be commercially available, suitable for transformation intoplants and suitable for maintenance and expression of a MAP gene in thetransformed cells. Preferably the genetic construct is a plantexpression vector.

The nucleic acid according to (a) is advantageously any of the nucleicacids described hereinbefore. A preferred nucleic acid is a nucleic acidrepresented by SEQ ID NO 1 or 3, or a variant thereof as hereinbeforedefined, or is a nucleic acid encoding a protein represented by SEQ IDNO 2 or 4, or a variant thereof as hereinbefore defined.

The terms “regulatory element” and “control sequence”, are used hereininterchangeably and are taken in a broad context refer to regulatorynucleic acid sequences capable of driving and/or regulating expressionof the sequences to which they are ligated and/or operably linked.Preferably, the control sequence of (b) is operable in a plant, mostpreferably the control sequence is a derived from a plant sequence.Encompassed by the terms “control sequence” are promoters. A “promoter”encompasses transcriptional regulatory sequences derived from aclassical eukaryotic genomic gene (including the TATA box which isrequired for accurate transcription initiation, with or without a CCAATbox sequence) and additional regulatory elements (i.e. upstreamactivating sequences, enhancers and silencers), which alter geneexpression in response to developmental and/or external stimuli, or in atissue-specific manner. Also included within the term is atranscriptional regulatory sequence of a classical prokaryotic gene, inwhich case it may include a -35 box sequence and/or -10 boxtranscriptional regulatory sequences. The term “regulatory element” alsoencompasses a synthetic fusion molecule or derivative, which confers,activates or enhances expression of a nucleic acid molecule in a cell,tissue or organ. The term “operably linked” as used herein refers to afunctional linkage between the promoter sequence and the gene ofinterest, such that the promoter sequence is able to initiatetranscription of the gene of interest. Preferably, the promoter isoperably linked to the gene of interest in a sense orientation.

Advantageously, any type of promoter may be used in the methods of thepresent invention. For example, a meristem-specific promoter, such asthe RNR (ribonucleotide reductase), cdc2a promoter and the cyc07promoter; or a seed-specific promoter, such as 2S2 albumin, prolamin, oroleosin promoter. A promoter may be selected in order to increaseexpression of a MAP protein at the moment of germination. Alternatively,a promoter expressed only in one or more of the seed tissues, such asthe aleurone, embryo, scutellum or endosperm may be used. Aflower-specific promoter, such as the leafy promoter, may be used if thedesired outcome would be to modify expression of a MAP gene in flowerorgans. An anther-specific promoter may be used to modify MAP expressionin male reproductive organs. Further, a root-specific promoter may beused, particularly in crops of which the roots are to be harvested; suchcrops include sugar beet, turnip, carrot, and potato. Avascular-specific promoter may be used or a nodule-specific promoter ora stress-inducible promoter. A cell wall-specific promoter may be usedor promoters expressed preferably in one or more of the above-groundtissues of the plant, such as green tissues, shoot, stem, leaves,fruits, and young expanding issues.

According to a preferred embodiment of the invention, the MAP nucleicacid in the genetic construct as described above, is operably linked toa constitutive promoter. The term “constitutive” as defined hereinrefers to a promoter that is expressed substantially continuously in andsubstantially in all tissues of a plant. Examples of useful constitutivepromoters are selected from the rice GOS2 promoter, maize GOS2 promoter,CaMV35S promoter, ubiquitin promoter, enolase promoter, actin-2 promoterand L-41 promoter or other promoters with similar expression patterns.Promoters with similar expression patterns may be found by coupling themto a reporter gene and checking the function of the reporter gene indifferent tissues of a plant. One suitable reporter gene isbeta-glucuronidase and the calorimetric GUS staining to visualize thebeta-glucuronidase activity in a plant tissue is well known to a personskilled in the art.

Optionally, one or more transcription termination sequences may also beincorporated in the genetic construct. The term “transcriptiontermination sequence” encompasses a control sequence at the end of atranscriptional unit, which signals 3′ processing and poly-adenylationof a primary transcript and termination of transcription. Additionalregulatory elements, such as transcriptional or translational enhancers,may be incorporated in the genetic construct. Those skilled in the artwill be aware of terminator and enhancer sequences, which may besuitable for use in the invention. Such sequences would be known or mayreadily be obtained by a person skilled in the art.

The genetic constructs of the invention may further include an origin ofreplication which is required for maintenance and/or replication in aspecific cell type. One example is when a genetic construct is requiredto be maintained in a bacterial cell as an episomal genetic element(e.g. plasmid or cosmid molecule) in a cell. Preferred origins ofreplication include, but are not limited to, f1-ori and colE1 ori.

The genetic construct may optionally comprise a selectable marker gene.As used herein, the term “selectable marker gene” includes any gene,which confers a phenotype on a cell in which it is expressed tofacilitate the identification and/or selection of cells, which aretransfected or transformed, with a genetic construct of the invention.Suitable markers may be selected from markers that confer antibiotic orherbicide resistance. Cells containing the recombinant DNA will thus beable to survive in the presence of antibiotic or herbicideconcentrations that kill untransformed cells. Examples of selectablemarker genes include genes conferring resistance to antibiotics (such asnptII encoding neomycin phosphotransferase capable of phosphorylatingneomycin and kanamycin, or hpt encoding hygromycin phosphotransferasecapable of phosphorylating hygromycin), to herbicides (for example barwhich provides resistance to Basta; aroA or gox providing resistanceagainst glyphosate), or genes that provide a metabolic trait (such asmanA that allows plants to use mannose as sole carbon source). Visualmarker genes result in the formation of colour (for examplebeta-glucuronidase, GUS), luminescence (such as luciferase) orfluorescence (Green Fluorescent Protein, GFP, and derivatives thereof).Further examples of suitable selectable marker genes include theampicillin resistance gene (Ampr), tetracycline resistance gene (Tcr),bacterial kanamycin resistance gene (Kanr), phosphinothricin resistancegene, and the chloramphenicol acetyltransferase (CAT) gene, amongstothers

According to another aspect of the present invention, there is provideda method for the production of plants, having modified growthcharacteristics relative to corresponding wild type plants, comprisingmodifying expression of a nucleic acid encoding a MAP protein and/ormodifying level and/or activity of a MAP protein in the plant. Accordingto one embodiment of the invention, such a method comprises introducinginto a plant cell a nucleic acid capable of modifying expression of anucleic acid encoding a MAP protein and/or capable of modifying leveland/or activity of a MAP protein.

According to a further embodiment of the invention, there is provided amethod for the production of plants having modified growthcharacteristics, which method comprises:

-   -   (a) Introducing into a plant cell a nucleic acid encoding a MAP        protein or variant thereof, or introducing a variant of a        nucleic acid encoding a MAP protein; and    -   (b) Cultivating said plant cell under conditions promoting plant        growth.

According to a further preferred embodiment, the nucleic acid of (a) isas represented by SEQ ID NO 1 or 2, or a variant thereof, or the nucleicacid of (a) encodes a MAP protein as represented by SEQ ID NO 2 or 4, ora variant thereof.

Cultivating the plant cell under conditions promoting plant growth, mayor may not include regeneration of the plant cell into a plant.Cultivating the plant cell under conditions promoting plant growth mayor may not include growth to reach maturity, including for example fruitproduction, seed formation, seed ripening and seed setting.

Methods for modifying expression of a MAP nucleic acid and/or formodifying level and/or activity of a MAP protein in a plant cell,include the introduction of the protein directly into said cell, forexample by microinjection or ballistic means. Alternatively, thesemethods include the introduction of a nucleic acid encoding a MAPprotein into a plant cell transient.

The MAP nucleic acid is preferably introduced into a plant bytransformation. The term “transformation” as referred to hereinencompasses the transfer of an exogenous polynucleotide into a hostcell, irrespective of the method used for transfer.

Plant tissue capable of subsequent clonal propagation, whether byorganogenesis or embryogenesis, may be transformed with a geneticconstruct of the present invention. The choice of tissue depends on theparticular plant species being transformed. Exemplary tissue targetsinclude leaf disks, pollen, embryos, cotyledons, hypocotyls,megagametophytes, callus tissue, existing meristematic tissue (e.g.,apical meristem, axillary buds, and root meristems), and inducedmeristem tissue (e.g., cotyledon meristem and hypocotyl meristem). Thenucleic acid may be transiently or stably introduced into a plant celland may be maintained non-integrated, for example, as a plasmid.Preferably, the nucleic acid encoding a MAP protein is stably introducedinto the genome of the transformed plant cell. Stable introduction intothe genome of a plant cell may be achieved, for example, by using aplant transformation vector or a plant expression vector having T-DNAborders which flank the nucleic acid to be introduced into the genome.

Transformation of a plant species is now a fairly routine technique.Advantageously, any of several transformation methods may be used tointroduce a MAP nucleic acid into a plant cell. Transformation methodsinclude the use of liposomes, electroporation, chemicals that increasefree DNA uptake, injection of the DNA directly into the plant, particlegun bombardment, transformation using viruses or pollen andmicroprojection. Methods may be selected from the calcium/polyethyleneglycol method for protoplasts (Krens, F. A. et al., 1882, Nature 296,72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373);electroporation of protoplasts (Shillito R. D. et al., 1985 Bio/Technol3, 1099-1102); microinjection into plant material (Crossway A. et al.,1986, Mol. Gen Genet 202, 179-185); DNA or RNA-coated particlebombardment (Klein T. M. et al., 1987, Nature 327, 70) infection with(non-integrative) viruses and the like. A preferred transformationmethod for the production of transgenic plants according to the presentinvention, is an Agrobacterium-mediated transformation method.

Transgenic rice plants are preferably produced viaAgrobacterium-mediated transformation using any of the well-knownmethods for rice transformation, such as the ones described in any ofthe following: published European patent application EP 1198985 A1,Aldemita and Hodges (Planta, 199, 612-617, 1998); Chan et al. (PlantMol. Biol. 22 (3) 491-506, 1993); Hiei et al. (Plant J. 6 (2) 271-282,1994); which disclosures are incorporated by reference herein as iffully set forth. In the case of corn transformation, the preferredmethod is as described in either Ishida et al. (Nat. Biotechnol. 1996June; 14(6): 745-0) or Frame et al. (Plant Physiol. 2002 May; 129(1):13-22), which disclosures are incorporated by reference herein as iffully set forth.

Generally after transformation, plant cells or cell groupings areselected for the presence of one or more of the above-mentionedselectable marker genes, which are co-transformed with the MAP gene.

The resulting transformed plant cells, cell groupings, or plant tissuemay than be used to regenerate a whole plant via techniques well knownto persons skilled in the art.

Subsequently, putatively transformed plant cells or plants may beevaluated, for instance using Southern analysis, for the presence of thegene of interest, copy number and/or genomic organisation. Alternativelyor additionally, expression levels of the introduced nucleic acid may beundertaken using northern and/or Western analysis, both techniques beingwell known to persons skilled in the art.

The regenerated transformed plants may be propagated by a variety ofmeans, such as by clonal propagation or classical breeding techniques.For example, a first generation transformed plant (T1 plant) may beselfed to give homozygous second generation transformed plants (T2plants), which T2 plants may be further propagated through classicalbreeding techniques.

The generated transformed plants may take a variety of forms. Forexample, they may be chimeras of transformed cells and non-transformedcells; clonal transformants (e.g., all cells transformed to contain thegenetic construct according to the invention); grafts of transformed anduntransformed tissues (e.g., a transformed rootstock grafted to anuntransformed scion).

The present invention clearly extends to plants obtainable by any of themethods according to the present invention, which plants have modifiedgrowth characteristics. The present invention dearly extends to anyplant part and propagules of such plants. The present invention extendsfurther to encompass the progeny of a primary transformed cell, tissue,organ or whole plant that has been produced by any of the aforementionedmethods, the only requirement being that progeny exhibit the samegenotypic and/or phenotypic characteristic(s) as the parent produced bythe methods according to the invention. The invention also includes hostcells having modified expression of a MAP gene and/or modified leveland/or activity of a MAP protein. Particularly, the invention includeshost cells comprising an isolated nucleic acid encoding a MAP protein.Such host cells preferably comprise a genetic construct as mentionedhereinabove. Preferred host cells according to the invention may beselected from bacteria, algae, fungi, yeast, insect plant or animal hostcells. The present invention extends to a transgenic plant cell or planthaving modified growth characteristics, which plant has modifiedexpression of a MAP gene and/or modified level and/or activity of a MAPprotein. Preferably said transgenic plant cell or plant comprising anisolated nucleic acid encoding a MAP protein or a variant thereof, morepreferably comprises a genetic construct as mentioned hereinabove. Theinvention also extends to any part of the plants according to theinvention, preferably a harvestable part, such as, but not limited to, aseed, leaf, fruit, flower, stem culture, stem, rhizome, root, tuber,bulb and cotton fiber.

The term “plant” or “plants” as used herein encompasses whole plants,ancestors and progeny of plants and plant parts, including seeds,shoots, stems, roots (including tubers), and plant cells, tissues andorgans. The term “plant” also therefore encompasses suspension cultures,embryos, meristematic regions, callus tissue, gametophytes, sporophytes,pollen, and microspores. Plants that are particularly useful in themethods of the invention include all plants which belong to thesuperfamily Viddiplantae, in particular monocotyledonous anddicotyledonous plants including a fodder or forage legume, ornamentalplant, food crop, tree, or shrub selected from the list comprisingAcacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathisaustralis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachisspp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaeaplunjuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkeaafricana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camelliasinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens,Chaenomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermummopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumisspp., Cupressus spp., Cyathea dealbata, Cydonia oblongs, Cryptomediajaponica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergiamonetada, Davallia divaricata, Desmodium spp., Dicksonia squarosa,Diheteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum,Echinochloa pyramidalis, Ehrartia spp., Eleusine coracana, Eragrestisspp., Erythina spp., Eucalyptus spp., Euclea schimperi, Eulalia villosa,Fagopyrum spp., Feijoa sellowiana, Fragana spp., Flemingia spp,Freycinetia banksii, Geranium thunbergii, Ginkgo biloba, Glycinejavanica, Glidcidia spp, Gossypium hirsutum, Grevillea spp., Guibourtiacoleosperma, Hedysarum spp., Hemarthia altissima, Heteropogon contortus,Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypertheliadissoluta, Indigo incarnate, Iris spp., Leptarrhena pyrolifolia,Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex,Lotonus bainesii, Lotus spp., Macrotyloma axillare, Malus spp., Manihotesculenta, Medicago sativa, Metasequoia glyptostroboides, Musasapientum, Nicotianum spp., Onobrychis spp., Omithopus spp., Oryza spp.,Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp.,Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp.,Picea glauca, Pinus spp., Pisum sativum, Podocarpus totara, Pogonarthriafleckii, Pogonarthria squarrosa, Populus spp., Prosopis cineraria,Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercusspp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis,Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubusspp., Salix spp., Schyzachyrium sanguineum, Sciadopitys verticillata,Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor,Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides,Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themedatriandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vacciniumspp., Vicia spp. Vitis vinifera, Watsonia pyramidata, Zantedeschiaaethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, brusselsprout, cabbage, canola, carrot, cauliflower, celery, collard greens,flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean,straw, sugarbeet, sugar cane, sunflower, tomato, squash, and tea, treesand algae amongst others.

According to a preferred embodiment of the present invention, the plantis a crop plant such as soybean, sunflower, canola, alfalfa, rapeseed,cotton, tomato, potato, tobacco, squash, papaya, poplar, leguminosa,flax, lupinus and sorghum. According to another preferred embodiment ofthe present invention, the plant is a monocotyledonous plant, such assugarcane, further preferably a cereal such as rice, maize (corn),wheat, barley, millet, rye and oats.

Accordingly, the present invention provides any of the methods asdescribed hereinabove, or a transgenic plant as described hereinabove,wherein the plant is a monocot, preferably a cereal, more preferablywherein the plant is rice or wherein the plant is corn.

Advantageously, performance of the methods according to the presentinvention leads to plants having various modified growthcharacteristics. The term “modified” as used herein means increased orimproved, decreased or changed over time and/or place. Preferably, withthe methods of the present invention the plant growth characteristicsare improved. The term “growth characteristic” as used herein,preferably refers to, but is not limited to yield/biomass and plantheight or any one or more of the growth characteristics as describedhereinbelow.

The term “yield” refers to the amount of harvestable material and isnormally defined as the measurable produce of economical value of acrop. For crop plants, “yield” also means the amount of harvestedmaterial per acre or unit of production. Yield may be defined in termsof quantity or quality. The harvested material may vary from crop tocrop, for example, it may be seeds (e.g. for rice, sorghum or corn whengrown for seed); aboveground biomass (e.g. for corn, when used assilage), roots (e.g. for sugar beet, turnip, potato), fruits (e.g. fortomato, papaya), cotton fibers, or any other part of the plant which isof economic value. “Yield” also encompasses yield stability of theplants. High yield stability means that year after year similar yieldfrom the progeny of the plants is obtained. “Yield” also encompassesyield potential, which is the maximum obtainable yield.

Yield may be dependent on a number of yield components, which may bemonitored by certain parameters. These parameters are well known topersons skilled in the art and vary from crop to crop. For example,breeders are well aware of the specific yield components and thecorresponding parameters for the crop they are aiming to improve. Forexample key yield parameters for corn include number of plants perhectare or acre, number of ears per plant, number of rows (of seeds) perear, number of kernels per row, and thousand kernel weight. For silagecorn, typical parameters are the above-ground biomass and energycontent. Key yield parameters for rice include number of plants perhectare or acre, number of panicles per plant, number of flowers(spikelets) per panicle, seed filling rate (number of filed seeds perspikelet) and thousand kernel weight. Preferentially methods forincreasing yield of rice encompass increasing number of filled seeds.

According to one particular embodiment, the term “yield” encompasses“seed yield”. The plants of the present invention are characterized byincreased harvested seed yield. The plants are characterized by anincreased number of filled seeds and increased total weight of harvestedseeds. Plants according to the present invention may also have increasedtotal number of seeds per plant. The methods of the present inventionare particularly favorable in cereals, such as rice and corn.Accordingly, a particular embodiment of the present invention relates toa method to increase yield of corn, for example seed yield, comprisingmodifying expression of a nucleic acid encoding a MAP protein.

The term “yield” also encompasses harvest index, which is the ratiobetween the harvested biomass over the total amount of biomass. Theplants of the present invention are characterized by increased harvestindex. Advantageously, the methods of the present invention may be usedto increase harvest index of cereals and in particular the harvest indexof corn or rice.

The term “yield” may also encompass Thousand Kernel Weight (TKW), whichis a parameter for the biomass of a single seed. Plants of the presentinvention may also exhibit increased TKW. Increased TKW is also anindication of increased seed volume and/or increased seed density.Advantageously, the methods of the present invention may be used toincrease the thousand kernel weight of cereals and in particular thethousand kernel weight of corn or rice may.

The term “yield” also encompasses typical biomass components, such asabove-ground biomass. General biomass parameters include above-groundarea and/or above-ground dry weight. The plants of the present inventionare characterized by increased above-round area. Therefore, the methodsof the present invention are particularly favorable for crops grown fortheir green issue and/or grown for their above-ground biomass. Themethods of the present invention are particularly useful for grasses,forage crops (such as forage corn, clover and medicago), trees and sugarcane.

The yield increase as obtained by the methods of the invention, mayresult from increase or improvement of one or more of the abovementioned yield components and/or parameters.

The term “growth characteristic” as used herein also encompasses plantheight. The plants according to the present invention may also exhibitincreased plant height.

The invention further relates to the use of an isolated nucleic acidencoding a MAP protein or use of an isolated MAP protein to modify plantgrowth characteristics. Furthermore, the invention embodies the use of aMAP gene or a MAP protein as a growth regulator, such as a herbicide ora growth stimulator. Also the present invention embodies a compositioncomprising a MAP nucleic acid or a MAP protein or a genetic construct asherein described above, for use as a regulator of plant growthcharacteristics (e.g. growth regulator such as a growth stimulator).Further, this composition comprises a suitable carrier, diluent orexcipient.

Alternatively, MAP genes and MAP proteins may be considered asinteresting targets for agrochemical compounds, such as a growthregulator or growth stimulator. Accordingly, the invention embodies theuse of a MAP gene or a MAP protein as a target of an agrochemicalcompound, such as or a growth regulator.

Since the plants of the present invention have excellent growthcharacteristics and have high yield, they are suitable for theproduction of enzymes, pharmaceuticals or agrochemicals. Also, they aresuitable for the production of food or feed products. The inventiondearly extends to enzymes, pharmaceuticals or agrochemicals as well asfood or feed products isolated or produced from these plants.

Furthermore, a nucleic acid encoding a MAP protein, a MAP protein and/orthe constructs of the present invention may be used in breeding programsaiming at the development of plants with increased yield. Further more,allelic variants as defined above may also be used in particularconventional breeding programs, such as in marker-assisted breeding.Such breeding programs sometimes need the introduction of allelicvariation in the plants by mutagenic treatment of a plant. One suitablemutagenic method is EMS mutagenesis. Identification of allelic variantsthen takes place by, for example, PCR. This is followed by a selectionstep for selection of superior allelic variants of the MAP sequence,which give rise to altered growth characteristics in a plant. Selectionis typically carried out by monitoring growth performance of plantscontaining different allelic variants of the MAP sequence, for exampledifferent allelic variants of the MAP sequence of SEQ ID NO 1.Monitoring growth performance can be done in a greenhouse and/or in thefield. Subsequently, plants having modified growth characteristics areselected. Such growth characteristics may be any one or more of thegrowth characteristics as described hereinabove. Further optional stepsinclude crossing the selected plants in which the superior allelicvariant was identified with another plant, for example a plant with aneconomically valuable genotype. These crossing methods may be used, forexample, to make a combination of interesting phenotypic features ortraits.

According to another type of breeding program, a DNA marker isidentified which may be genetically linked to the gene capable ofmodifying expression of a MAP gene and/or modifying level and/oractivity of a MAP protein in a plant (which gene may be the geneencoding the MAP protein itself or another gene capable of modifyingexpression of a MAP gene or capable of modifying level and/or activityof the MAP protein). This DNA marker is then used in breeding programsto select plants having modified growth characteristics. Such growthcharacteristics may be any one or more of the growth characteristics asdescribed hereinabove.

The methods according to the present invention may also be practised byintroducing into a plant at least a part of a (natural or artificial)chromosome (such as a Bacterial Artificial Chromosome (BAC)), whichchromosome contains at least a gene encoding a MAP protein, optionallytogether with one or more related gene family members. Therefore,according to a further aspect of the present invention, there isprovided a method for modifying plant growth characteristics comprisingintroducing into a plant at least a part of a chromosome comprising atleast a gene encoding a MAP protein.

The present invention will now be described with reference to thefollowing figures in which:

FIG. 1 is a map of the plant expression vector comprising an expressioncassette for a MAP gene under control of a constitutive promoter.CDS0430 is the internal code for the Arabidopsis thaliana cDNA encodingmethionine aminopeptidase MAP2B. PRO0129 is the internal code for therice GOS2 promoter. The MAP expression cassette also comprises thedouble transcription termination sequence T-zein and T-rbcS-deltaGA.This expression cassette is located within the left border (LB Ti C58)and the right border (RB Ti C58) of the nopaline Ti plasmid. Clonedwithin these T-borders are also a screenable marker and a selectablemarker both under control of a constitutive promoter and followed by aNOS transcription termination sequence. Furthermore, this vector alsocontains an origin of replication (pBR322 (ori+bom) for bacterialreplication and a bacterial selectable marker (Sm/SpR) for bacterialselection.

FIG. 2 lists all the sequences used in the description of the presentinvention

FIG. 3 shows a multiple alignment of animal and plant MAP proteins, withthe annotation of the different domains. From the N-terminus to theC-terminus: lysine-rich domain, MAP2 signature or MAP1 signature andPeptidase_M24 domain. The peptidase domain is annotated corresponding tothe peptidase domain of MAP2 proteins (see for example SEQ ID NO 8). Thepeptidase domain of MAP1 proteins extends further than this annotation(see for example SEQ ID NO 7).

EXAMPLES

The present invention will now be described with reference to thefollowing examples, which are by way of illustration alone.

DNA Manipulation

Unless otherwise stated, recombinant DNA techniques are performedaccording to standard protocols described in Sambrook (2001) MolecularCloning: a laboratory manual, 3rd Edition Cold Spring Harbor LaboratoryPress, CSH, New York or in Volumes 1 and 2 of Ausubel et al. (1988),Current Protocols in Molecular Biology, Current Protocols. Standardmaterials and methods for plant molecular work are described in PlantMolecular Biology Labfase (1993) by R. D. D. Croy, published by BIOSScientific Publications Ltd (UK) and Blackwell Scientific Publications(UK).

Example 1 Cloning of CDS0430 Encoding Arabidopsis thaliana MAP2B

A gene encoding a methionine aminopeptidase MAP2B was amplified by PCRfrom an Arabidopsis thaliana seedling cDNA library (Invitrogen, Paisley,UK). After reverse transcription of RNA extracted from seedlings, thecDNAs were cloned into pCMV Sport 6.0. Average insert size of the bankwas about 1.5 kb, and original number of clones was about 1.59×10⁷ cfu.Original titre was determined to be 9.6×10⁵ cfu/ml, after firstamplification of 6×10¹¹ cfu/ml. After plasmid extraction, 200 ng oftemplate was used in a 50 μl PCR mix. The primers used for PCRamplification, prm01642 with the sequence 5′ACAAGTTTGTACAAAAAAGCAGGCTTCA CAATGGCGAGCGAAAGTCC 3′ as represented bySEQ ID NO 10 and prm01643 with the sequence 5′ACCCAGCTTTCTTGTACAAAGTGGTA GGATCTGMTCAGTAGTCGTCTC 3′ as represented bySEQ ID NO 11, include an attB site for Gateway recombination (italics).PCR was performed using Hifi Taq DNA polymerase in standard conditions.A PCR fragment of 1381 bp was amplified and purified using standardmethods. The first step of the Gateway procedure, the BP reaction, wasthen performed, during which the PCR fragment recombines in vivo withthe pDONR201 plasmid to produce the entry done p1753. Plasmid pDONR201was purchased from Invitrogen, as part of the Gateway) technology.

Example 2 Construction of Expression Cassette CD2231 (pGOS2::AtMAP2B)

The entry clone p1753 was subsequently used in an LR Gatewayrecombination reaction with p0640, a Gateway destination vector suitablefor rice transformation. Vector p0640 contains as functional elementswithin the T-DNA borders, a plant selectable marker and a Gatewaycassette intended for LR in vivo recombination with the sequence ofinterest already cloned in the entry clone. Upstream of this Gatewaycassette lies the rice GOS2 promoter (PRO0129) for constitutiveexpression of the gene of interest (De Pater et al., Plant J. 2(6)837-844, 1992). After the recombination step, the resulting expressionvector with the expression cassette CD2231 (FIG. 1) can be transformedinto Agrobacterium strain LBA4404 and subsequently into Oryza sativavar. Nipponbare plants. Transformed rice plants were allowed to grownand were examined for various growth characteristics as described inExample 3.

Example 3 Evaluation of T0, T1 and T2 Rice Plants Transformed withpGOS2::AtMAP2B

Approximately 15 to 20 Independent T0 transformants were generated. Theprimary transformants were transferred from tissue culture chambers to agreenhouse for growing and harvest of T1 seed. Six events of which theT1 progeny segregated 3/1 for presence/absence of the transgene wereretained. “Null plants” or “Null segregants” or “Nullizygotes” are theplants treated in the same way as a transgenic plant, but from which thetransgene has segregated. Null plants can also be described as thehomozygous negative transformants. For each of these events,approximately 10 T1 seedlings containing the transgene (hetero- andhomo-zygotes), and approximately 10 T1 seedlings lacking the transgene(nullizygotes), were selected by PCR.

Based on the results of the T1 evaluation, three events, which showedimproved growth characteristics at the T1 level, were chosen for furthercharacterisation in the T2 and further generations. To this extent, seedbatches from the positive T1 plants (both hetero- and homozygotes), werescreened by monitoring marker expression. For each chosen event, theheterozygote seed batches were then selected for T2 evaluation. An equalnumber of positive and negative within each seed batch were transplantedfor evaluation in the greenhouse (i.e., for each event 40 plants, ofwhich 20 positives for the transgene and 20 negative for the transgene,were grown). For the three events therefore, a total amount of 120plants was evaluated in the T2 generation.

T1 and T2 plants were transferred to a greenhouse and were evaluated forvegetative growth parameters and seed parameters, as describedhereunder.

(I) Statistical Analysis of Numeric Data

A two factor ANOVA (analyses of variance) corrected for the unbalanceddesign was used as statistical evaluation model for the numeric valuesof the observed plant phenotypic characteristics. The numerical valuesare submitted to a t-test and an F test. The p-value is obtained bycomparing the t value to the t distribution or alternatively, bycomparing the F value to the F distribution. The p-value stands theprobability that the null hypothesis (null hypothesis being “there is noeffect of the transgene”) is correct.

A t-test was performed on all the values of all plants of one event.Such a t-test was repeated for each event and for each growthcharacteristic. The t-test was carried out to check for an effect of thegene within one transformation event, also named herein a “line-specificeffect”. In the t-test, the threshold for a significant line-specificeffect is set at 10% probability level. Therefore, data with a p-valueof the t test under 10% indicate a “line-specific” effect, meaning thatthe phenotype observed in the transgenic plants of that line is causedby the presence of the gene. Within one population of transformationevents, some events may be under or below his threshold. This differencemay be due to the difference in position of the transgene in the genome.It is not uncommon that a gene might only have an effect in certainpositions of the genome. Therefore, the above-mentioned “line-specificeffect” is also referred to ad “position-dependent effect”.

An F-test was carried out on all the values measured for all plants ofall events. An F-test was repeated for each growth characteristic. TheF-test was carried out to check for an effect of the gene over all thetransformation events and to verify an overall effect of the gene, alsonamed herein “gene effect”. In the F-test, the threshold for asignificant global gene effect is set at 5% probability level.Therefore, data with a p-value of the F test under 5% indicate a “geneeffect”, meaning that the phenotype observed is caused by more than justthe presence of the gene and or the position of the transgene in thegenome. A “gene effect” is an indication for the wide applicability ofthe gene in transgenic plants.

(II) Vegetative Growth Measurements

The selected plants were grown in a greenhouse. Each plant received aunique barcode label to link unambiguously the phenotyping data to thecorresponding plant. The selected plants were grown on soil in 10 cmdiameter pots under the following environmental settings:photoperiod=11.5 h, daylight intensity=30,000 lux or more, daytimetemperature=28° C. or higher, night time temperature=22° C., relativehumidity=60-70%. Transgenic plants and the corresponding nullizygoteswere grown side-by-side at random positions. From the stage of sowinguntil the stage of maturity (which is the stage were there is no moreincrease in biomass) the plants were passed weekly through a digitalimaging cabinet. At each time point digital images (2048×1536 pixels, 16million colours) were taken of each plant from at least 6 differentangles. The parameters described below were derived in an automated wayfrom the digital images using image analysis software.

(a) Aboveground Area

Plant above-ground area was determined by counting the total number ofpixels from above-ground plant parts discriminated from the background.This value was averaged for the pictures taken on the same time pointfrom the different angles and was converted to a physical surface valueexpressed in square mm by calibration. Experiments show that theabove-ground plant area, which corresponds to the total maximum area,measured this way correlates with the biomass of plant partsabove-ground.

These results of the maximum above-ground area are summarized inTable 1. On average, transgenic plants show an increase in above-groundarea of 10%. In one particular line the increase in above-ground areawas as high as 26%. These results indicate that the MAP gene has aneffect on the above-ground biomass of MAP transgenic plants.

Table 1: Maximum above-ground area of MAP transgenic T2 plants. Each rowcorresponds to one event, for which the average maximum above-groundarea (expressed in mm²) was determined for the transgenics (TR) and thenull plants (null). The difference between the transgenic plants and thenull plants of each event is presented in absolute values (dif.), aswell as in percentage of difference (% dif). P stands for theprobability produced by the t-test for each event. The last row presentsthe average numbers calculated from all the events. Here the p-value isproduced by the F-test. Maximum above-ground area Line TR null dif % difp-value CD2231 L1 67117 66542 576 1 0.9193 CD2231 L2 51901 49645 2257 50.6912 CD2231 L3 77716 61626 16090 26 0.0065 Overall 65647 59498 6149 100.0495(III) Measurement of Seed-Related Parameters

The mature primary panicles were harvested, bagged, barcode-labelled andthen dried for three days in the oven at 37° C. The panicles were thenthreshed and all the seeds collected. The filled husks were separatedfrom the empty ones using an air-blowing device. After separation, bothseed lots were then counted using a commercially available countingmachine. The empty husks were discarded. The filled husks were weighedon an analytical balance. This procedure resulted in the set ofseed-related parameters described below.

(a) Total Number of Filled Seeds per Plant

The number of filled seeds was determined by counting the number offilled husks that remained after the separation step. These numbers aresummarized In Table 2. On average, transgenic plants show an increase innumber of filled seeds of 52%. In one particular line the increase innumber of filled seeds was as high as 76%. These results indicate thatthe MAP gene has an effect on the number of filled seeds of MAPtransgenic plants.

Table 2: Number of filled seeds of MAP transgenic 72 plants. Each rowcorresponds to one event, for which number of filled seeds wasdetermined for the transgenics (TR) and the null plants (null). Thedifference between the transgenic plants and the null plants of eachevent is presented in absolute values (dif.), as well as in percentageof difference (% dif). P stands for the probability produced by thet-test for each event. The last row presents the average numberscalculated from all the events. Here the p-value is produced by theF-test. Number of filled seeds Line TR null dif % dif p-value CD2231 L1328.2 216 112.21 52 0.0132 CD2231 L2 157.1 137.2 19.91 15 0.6551 CD2231L3 412 233.8 178.21 76 0.0001 Overall 300.5 198.1 102.36 52 <0.0001(b) Total Seed Yield per Plant

The total seed yield was measured as total seed weight, by weighing allfilled husks harvested from a plant. The values of total seed weight aresummarized in Table 3. On average, transgenic plants show an increase intotal seed weight of 55%. In one particular line the increase in totalseed weight and thus in seed yield was as high as 79%. These resultsindicate that the MAP gene has an effect on the total seed weight andseed yield of MAP transgenic plants.

Table 3: Total seed weight per plant of MAP transgenic T2 plants. Eachrow corresponds to one event, for which the average total seed weight(in grams) was determined for the transgenics (TR) and the null plants(null). The difference between the transgenic plants and the null plantsof each event is presented in absolute values (dif.), as well as inpercentage of difference (% dif). P stands for the probability producedby the f-test for each event. The last row presents the average numberscalculated for all the events. Here the p-value is produced by theF-test. Total seed weight Line TR null dif % dif p-value CD2231 L1 9 5.93.08 52 0.0071 CD2231 L2 3.7 3.1 0.63 20 0.5746 CD2231 L3 11.1 6.2 4.979 <0.0001 Overall 8 5.1 2.83 55 <0.0001(c) Harvest Index

The harvest index in the present invention is defined as the ratiobetween the total seed yield and the above-ground area (mm²), multipliedby a factor 10⁶. The values for harvest index are summarized in Table 4.On average, transgenic plants show an increase in harvest index of 36%.In one particular line the increase in harvest index and thus theincrease in yield was as high as 50%. These results indicate that theMAP gene has an effect on the harvest index and on yield of the MAPtransgenic plants.

Table 4: Harvest index of MAP transgenic T2 plants. Each row correspondsto one event, for which the average harvest index was determined for thetransgenics (TR) and the null plants (null). The difference between thetransgenic plants and the null plants of each event is presented inabsolute values (dif.), as well as in percentage of difference (% dif).P stands for the probability produced by the t-test for each event. Thelast row presents the average numbers calculated from all the events.Here the p-value is produced by the F-test. Harvest Index Line TR nulldif % dif p-value CD2231 L1 126.4 84.1 42.24 50 0.0002 CD2231 L2 67.459.8 7.63 13 0.4843 CD2231 L3 138.6 99.1 39.52 40 0.0007 Overall 110.580.9 29.53 36 <0.0001(d) Thousand Kernel Weight

TKW is extrapolated from the number of filled seeds counted and theirtotal weight. Some MAP transgenic plants showed an increase in Thousandkernel weight, which is an indicator of increased seed density and/orincreased seed size.

(IV) Measurement of Plant Height

Plant height was determined by the distance between the horizontal linesgoing through the upper pot edge and the uppermost pixel correspondingto a plant part above ground. This value was averaged for the picturestaken on the same time point from the different angles (taken asdescribed above) and was converted, by calibration, to a physicaldistance expressed in mm. Experiments showed that plant height measuredthis way correlate with plant height measured manually with a ruler.Some MAP transgenic plants showed an increase of plant height.

Example 4 Evaluation of Transgenic Corn Plants Transformed with MAP

The methods of the invention described herein are also used in corn (Zeamays). To this aim, a MAP encoding gene, for example a corn MAPorthologue, is cloned under control of a promoter operable in corn, in aplant transformation vector suitable for Agrobacterium-mediated corntransformation. The promoter operable in corn may for example be aconstitutive promoter, selected from GOS2 promoters, ubiquitinepromoters, L41 promoters, actine-2 promoters and enolase promoters.Methods to use for corn transformation have been described in literature(Ishida et al., Nat Biotechnol. 1996 June; 14(6):745-50; Frame et al.,Plant Physiol. 2002 May; 129(1):13-22).

Transgenic (inbred) lines made by these methods may be crossed withanother non-transgenic or transgenic (inbred) line or beself/sib-pollinated. Importantly, transgenic (inbred) lines may be usedas a female or male parent inheritability and copy number of thetransgene are checked by quantitative real-time PCR and Southern blotanalysis and expression levels of the transgene are determined byreverse PCR and Northern analysis. Transgenic events with single copyinsertions of the transgene and with varying levels of transgeneexpression are selected for further evaluations in subsequentgenerations.

Progeny seeds obtained as described hereinabove are germinated and grownin the greenhouse in conditions well adapted for corn (16:8 photoperiod,26-28° C. daytime temperature and 20-24° C. night time temperature) aswell under water-deficient, nitrogen-deficient, and excess NaClconditions. Null segregants from the same parental line (inbred line orhybrids), as well as wild type plants of the same inbred line or hybridsare used as controls. The progeny plants are evaluated on differentbiomass and developmental parameters, including but not limited to plantheight, stalk width, nodes below ear, nodes above ear, brace roots,number of leaves, leaf greenness, leaf angle, total above-ground areatime to tassel, time to silk, time to maturity, ear height, ear number,ear length, ear weight, row number, kernel number, grain moisture.Kernel traits include but are not limited to kernel size, kernel weightstanch content, protein content, and oil content are also monitored.Corn yield is calculated according to well-known methods. Corn plantstransformed with a MAP protein show improved growth characteristics.More particularly they show an improvement in any one or more of theabovementioned biomass and developmental parameters.

Transgenic events that are most significantly improved compared tocorresponding control lines are selected for further field-testing andmarker-assisted breeding, with the objective of transferring thefield-validated transgenic traits into another germplasm. Thephenotyping of maize for growth and yield-related parameters in thefield is conducted using well-established protocols. The corn plants areparticularly evaluated on yield components at different plant densitiesand under different environmental conditions. Subsequent improvementsfor introgressing specific loci (such as transgene containing loci) fromone germplasm into another is also conducted using well-establishedprotocols including but not limited to MAS.

Example 5 Evaluation of Transgenic Plants Transformed with Arabidopsisthaliana MAP1A

The methods described in Examples 1, 2, 3 and 4 are also repeated withArabidopsis thaliana MAP1A. To this aim, the AtMAP1A encoding gene,represented by SEQ ID NO 3, is cloned under control of a promoteroperable in rice or maize, in a plant transformation vector suitable forAgrobacterium-mediated transformation of rice or corn. One suitablepromoter is a constitutive promoter, such as a GOS2 promoter.

In case of rice plants, the protocol for isolation of cDNA and forvector construction is followed as described in Example 1 and 2, exceptthat the primers are specific for SEQ ID NO 3. The protocols for planttransformation, plant growth and plant evaluation are followed asdescribed in Example 3. Rice plants transformed with AtMAP1A havemodified growth characteristics and show any one or more of the modifiedgrowth characteristics as described in Example 3.

In case of corn plants, the protocols for plant transformation, plantgrowth, plant propagation, plant selection, and plant evaluation arefollowed as described in Example 4. Corn plants transformed with AtMAP1Ahave modified growth characteristics and show any one or more of theimproved growth characteristics as described in Example 4.

1. Method for modifying plant growth characteristics relative tocorresponding wild type plants, comprising modifying expression in aplant of a nucleic acid encoding a methionine aminopeptidase (MAPprotein) and/or modifying level and/or activity in a plant of a MAPprotein.
 2. Method according to claim 1, wherein said modifyingexpression, level and/or activity is effected by introducing into aplant a nucleic acid capable of modifying expression of a nucleic acidencoding a MAP protein and/or capable of modifying level and/or activityof a MAP protein.
 3. Method according to claim 2, wherein the introducednucleic acid encodes a MAP protein or a variant thereof and/or whereinsaid introduced nucleic acid is a variant of a nucleic acid encoding aMAP protein.
 4. Method according to claim 1, wherein said modifyingexpression, level and/or activity comprises increased expression, leveland/or activity.
 5. Method for the production of a plant having modifiedgrowth characteristics relative to corresponding wild type plants,comprising: a) Introducing into a plant cell a nucleic acid encoding aMAP protein or a variant thereof, or introducing a variant of a nucleicacid encoding a MAP protein; and b) Cultivating said plant cell underconditions promoting plant growth.
 6. Method according to claim 5,wherein the nucleic acid of (a) is as represented by SEQ ID NO 1 or 3,or a variant thereof, or wherein said nucleic acid of (a) encodes a MAPprotein as represented by SEQ ID NO 2 or 4, or a variant thereof. 7.Method according to claim 5, wherein said nucleic acid encodes ahomologue having, in increasing order of preference, at least 35%, 36%,37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%,80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98% or 99% sequenceidentity with the sequence of SEQ ID NO 2 or with the sequence of SEQ IDNO
 4. 8. Method according to claim 5, wherein said nucleic acid encodinga MAP protein is obtained from a plant.
 9. Method according to claim 1,wherein said modified growth characteristic is increased yield. 10.Method according to claim 9, wherein said modified growth characteristicis increased seed yield.
 11. Method according to claim 1, wherein saidgrowth characteristic is increased plant height.
 12. Genetic constructcomprising: a) A nucleic acid encoding a plant MAP protein or a variantthereof, or comprising a variant of a nucleic acid encoding a plant MAPprotein; and b) One or more control sequence capable of drivingexpression of the nucleic acid of (a) in a plant; and optionally c) Atranscription termination sequence.
 13. Genetic construct according toclaim 12, wherein the nucleic acid of (a) is as presented in SEQ ID NO 1or 3, or a variant thereof, or wherein said nucleic acid of (a) encodesa MAP protein as presented in SEQ ID NO 2 or 4, or a variant thereof.14. Genetic construct according to claim 12, wherein said nucleic acidof (a) encodes a homologue having, in increasing order of preference, atleast 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%,48%, 49%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%,74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 99%sequence identity with the sequence of SEQ ID NO 2 or with the sequenceof SEQ ID NO
 4. 15. Genetic construct according to claim 12, whereinsaid nucleic acid of (a) is as presented in SEQ ID NO 1 or SEQ ID NO 3.16. Genetic construct according to claim 12, wherein said controlsequence of (b), is a constitutive promoter, such as a GOS2 promoter.17. Host cell containing a genetic construct as defined in claim
 12. 18.Plant obtainable by a method according to claim 1, which plant hasmodified growth characteristics relative to corresponding wild typeplants.
 19. Transgenic plant having modified growth characteristics,which plant has modified expression of a nucleic acid encoding a MAPprotein, and/or has modified level and/or activity of a MAP protein,relative to corresponding wild type plants.
 20. Transgenic plantaccording to claim 19, containing an isolated nucleic acid encoding aMAP protein or a variant thereof, or containing an isolated variant of anucleic acid encoding a MAP protein, or containing a genetic constructas defined in any of claims 12 to
 16. 21. Plant according to claim 20,which plant is a monocotyledonous plant, preferably a cereal, such ascorn or rice.
 22. Plant according to claim 18, wherein said growthcharacteristic is increased yield.
 23. Plant according to claim 22,wherein said increased yield is increased seed yield.
 24. Plantaccording to claim 18, wherein said growth characteristic is increasedplant height.
 25. Plant part, preferably a harvestable part such as aseed, a propagule or progeny of a plant as defined in claim
 18. 26. Useof an isolated nucleic acid encoding a MAP protein or use of an isolatedMAP protein to modify plant growth characteristics.
 27. Use according toclaim 26, wherein said growth characteristic is increased yield.
 28. Useaccording to claim 27, wherein said increased yield is increased seedyield.
 29. Use according to claim 26, wherein said growth characteristicis increased plant height.
 30. Composition suitable for regulation ofplant growth characteristics, comprising a nucleic acid encoding a MAPprotein, a MAP protein or a genetic construct as defined in claim 12,and further comprising a suitable carrier, diluent or excipient.
 31. Useof a nucleic acid encoding a MAP protein or use of a MAP protein as atarget for a plant growth regulator.
 32. Use of a nucleic acid encodinga MAP protein in a plant breeding program aimed at improving yield. 33.Use of a plant as defined in claim 18 or of a plant part as defined inclaim 25, in the production of enzymes, pharmaceuticals oragrochemicals.
 34. Use of a plant as defined in claim 18 or of a plantpart as defined in claim 25, in the production of food or feed products.